Next Article in Journal
Changes in Vertical Jump Parameters After Training Unit in Relation to ACE, ACTN3, PPARA, HIF1A, and AMPD1 Gene Polymorphisms in Volleyball and Basketball Players
Next Article in Special Issue
Phylogeny and Specific Determination of Gloydius halys-intermedius Complex Based on Complete Mitochondrial Genes
Previous Article in Journal
The Association Between Statin Drugs and Rhabdomyolysis: An Analysis of FDA Adverse Event Reporting System (FAERS) Data and Transcriptomic Profiles
Previous Article in Special Issue
Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 16
Issue 3
10.3390/genes16030249
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification, Gene Duplication, and Expression Pattern of NPC2 Gene Family in Parnassius glacialis

by
Zhenyao Zhu
†,
Chengyong Su
†,
Xuejie Guo
,
Youjie Zhao
,
Ruie Nie
,
Bo He
and
Jiasheng Hao
*
College of Life Sciences, Anhui Normal University, Wuhu 241000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2025, 16(3), 249; https://doi.org/10.3390/genes16030249
Submission received: 25 January 2025 / Revised: 14 February 2025 / Accepted: 20 February 2025 / Published: 21 February 2025
(This article belongs to the Special Issue Molecular Evolution, Mitochondrial Genomics and Mitochondrial Genome Expression in Animals: 2024–2025)
Browse Figures
Versions Notes

Abstract

:
Background: The Niemann–Pick C2 (NPC2) gene family plays an important role in olfactory communication, immune response, and the recognition of host plants associated with environmental adaptation for insects. Methods: In this study, we conducted a genomic analysis of the structural characteristics and physicochemical properties of the NPC2 genes of eleven butterfly species available, focusing on the alpine Parnassius species, especially Parnassius glacialis, to investigate their duplication and expression patterns. Results: Our results indicate that a significant expansion of NPC2 genes was detected in P. glacialis compared to other butterflies; in addition, the expansion of these unevenly distributed P. glacialis NPC2 chromosome genes was shaped by tandem duplication mediated by transposons. Furthermore, the PgNPC2 genes had relatively higher expression in P. glacialis antennae and other head tissues. These facts were verified by quantitative real-time PCR (qRT-PCR). Conclusions: These findings suggest that the expansion of NPC2 genes may have contributed to the local adaptation of P. glacialis during its dispersal ‘out of the Qinghai–Tibet Plateau’, although further functional tests are needed to confirm their specific role in this adaptive process.

1. Introduction

The biological diversity pattern, especially in the Northern Hemisphere, has been significantly influenced by the geological events and climate changes in the Qinghai–Tibet Plateau (QTP) since the early Cenozoic era [1,2,3]. Among insect groups that originated on the QTP, the genus Parnassius is a typical high mountain-adapted butterfly group that is mainly distributed across the Holarctic region, especially on the QTP and neighboring mountains with an elevation ranging from about 3000 m to 5000 m. Previous studies have shown that the Parnassius probably originated in the early to middle Miocene about 20 to 15 million years ago (mya), and since that period, they have undergone rapid adaptive radiations and local specializations due to topographical isolation and habitat fragmentation events caused by the QTP uplifting, as well as climate changes, especially the glacial–interglacial cycles in the Quaternary Period [1,2,3]. Thus, this typical alpine butterfly group has emerged as an ideal model for studying the relationship between biological evolution and the earth’s environmental change, such as phylogenetics, phylogeography, and other areas of the earth–life system [4,5,6].
P. glacialis is the only Parnassius species that dispersed eastwards from the QTP to southern China (south of Yangtze River) and inhabits areas at altitudes ranging from about 2800 m to 300 m. Based on the genomic analysis of previous studies, this species began to disperse at about 1.1 to 0.6 mya, which coincides closely with the Kunlun-Huanghe tectonic movement [4,5,6,7], and during its dispersal processes, it underwent a rapid and extensive expansion of gene families (about 700), especially the RPLP2 gene family, which contains about 434 genes [4,8,9]. Except for one gene that is structurally and functionally normal, these RPLP2 pseudogenes are somewhat newly functional, and their population is characteristically expressed, which could contribute to their local adaptation to different environments and to the formation of some evolutionary traits, such as a larger body size [4]. In addition, these gene expansions are all driven by LTR transposons [8]. However, to date, little attention has been paid to other gene families that are associated with the genomic differentiation contributing to the local adaptation of this alpine butterfly species.
The Niemann–Pick C2 (NPC2) gene, characterized by the presence of the MD-2-related lipid recognition (ML) domain, was firstly reported and clarified as essential for lipid metabolism and cholesterol transport in vertebrates [10,11,12,13,14]. Nowadays, increasing evidence has shown that multiple NPC2 genes are responsible for the regulation of olfactory communication, steroidogenesis, immune response, mating, and reproduction in some insect groups [15,16,17,18]. For example, eight NPC2 genes (NPC2a-h) were identified in Drosophila for regulating steroidogenesis, growth, and molting by controlling sterol homeostasis and steroid biosynthesis [16]; furthermore, NPC2e and NPC2a were detected to bind bacterial cell wall components and function in immune signal pathways, respectively, in Drosophila melanogaster [18]. NPC2 genes have also been shown to promote olfactory communication in the ant Camponotus japonicas [17], to help in the recognition of host plants in the honeybee Apis cerana [15], and in the mating and reproduction processes in the parasitoid wasp Microplitis mediator [19].
In this study, we attempted to conduct a comprehensive analysis of the NPC2 gene family regarding their gene numbers, their correspondent protein physicochemical properties, and their gene structures and gene duplication mechanisms in 11 available butterfly species, focusing on the Parnassius, especially the P. glacialis. In addition, we analyzed the NPC2 cis-acting elements to explore their regulatory mechanisms and the NPC2 gene expression levels in different P. glacialis tissues through quantitative real-time PCR (qRT-PCR) to clarify their tissue-characteristic expressions. The results will deepen our understanding of the Parnassius NPC2 gene family and provide some new insights into their local adaptation mechanisms.

2. Materials and Methods

2.1. The Identification of the NPC2 Gene Family

All genomic sequences and annotation files of 11 butterfly species, including the P. glacialis, were obtained based on two main criteria: phylogenetic representation across different butterfly families and genome availability. These species include P. glacialis and the Parnassius apollo [20] (GCA_907164705.1), the P. orleans [21] (GCA_029286625.1), the Papilio machaon (GCA_001298355.1), the Papilio bianor [22], the Pieris napi (GCA_905475465.1), the Pieris rapae (GCA_905147795.1), the Fabriciana adippe (GCA_905404265.1), the Hesperia comma (GCA_905404135.1), Celastrina argiolus (GCA_905187575.1), and Heliconius erato [23], which were obtained from the public NCBI (https://www.ncbi.nlm.nih.gov/genome, accessed on 14 August 2023), GigaDB (https://gigadb.org/dataset/100653, accessed on 14 August 2023) and LepBase (http://butterflygenome.org/?q=node/4/H.erato ltivitta v1.0, accessed on 14 August 2023) databases, respectively. The corresponding NPC2 protein sequences of Drosophila melanogaster (NP_731880.1) were downloaded from the GenBank of the NCBI (National Center for Biotechnology Information) database (https://www.ncbi.nlm.nih.gov/protein, accessed on 14 August 2023) for reference sequences; in order to identify the candidate P. glacialis NPC2 proteins, an analysis of a chromosome-level genome that was previously determined by our laboratory was performed using TBtools-II v2.154 [24] with the e-value threshold of 1 × 10−5. Additionally, the Hidden Markov Model (HMM) file of the NPC2 DNA binding domain (PF02221) from the Pfam database (http://pfam.xfam.org/, accessed on 27 August 2023) was used to further identify the candidate NPC2 gene sequences. Then, all of the above candidate gene sequences were verified using the NCBI-BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome, accessed on 12 September 2023) and NCBI Batch-CDD search (https://structure.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 16 September 2023) methods. Finally, these verified genes of the NPC2 family were renamed according to their locations on the chromosomes.

2.2. Analysis of Physicochemical Properties, Secondary Structure, and Subcellular Localization

The number of amino acids, molecular weight, isoelectric point, hydrophobic index, and other physicochemical properties of the above selected NPC2 members were analyzed online using ExPASy (https://web.expasy.org/protparam, accessed on 28 September 2023). The secondary structures and the subcellular localizations of these NPC2 proteins were predicted using the online tools Network Protein Sequence analysis (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 12 October 2023) and WoLF PSORT (https://wolfpsort.hgc.jp, accessed on 16 October 2023), respectively [25].

2.3. Analysis of Multiple Sequence Alignment and Phylogenetic

In order to explore the evolutionary relationship of these NPC2 genes in butterflies, firstly, all NPC2 sequences of the 11 butterfly species were retrieved from the genomic databases mentioned above; secondly multiple sequence alignment was performed, and the results were further processed using the Muscle program of the MEGA software (version 11) [26], and TrimAL 2.0 software [27], respectively; lastly, the best NPC2 phylogenetic tree of the 11 butterfly species was reconstructed with the neighbor-joining method through 1000 bootstrap replicates using the MEGA software, and the evolutionary tree was visualized and beautified by the online software iTOL (https://itol.embl.de/, accessed on 19 August 2024) [28].

2.4. An Analysis of the Conserved Motifs, Gene Structures, and Characteristic Domains

The NPC2 conserved motif was predicted using the MEME Suite v5.5.4 software (https://meme-suite.org/meme/, accessed on 2 April 2024) [29,30] with the E-value threshold of <0.05 and the following parameters: the minimum width (6 bp), maximum width (25 bp), and the maximum number of motifs (15). The NPC2 exon–intron structure was retrieved from the GFF3 (General Feature Format 3) annotation file. The NPC2 characteristic domain was identified using the NCBI website (https://structure.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 6 May 2024). Subsequently, the conserved motif, gene structure, and the characteristic domain of the NPC2 genes were visualized using TBtools-II v2.154 [24].

2.5. An Analysis of the Chromosome Distribution, Collinearity, and Gene Duplication

To explore the chromosomal locations of NPC2 genes of the three Parnassius butterfly species, the NPC2 gene loci were retrieved from genomic files [24]. Both the gene duplication and collinearity analyses were performed using MCScanX in TBtools software with default parameters to investigate the evolutionary relationship of the NPC2 genes in Parnassius species and two other Papilionidae species (Pa. bianor and Pa. machaon) [31,32,33]. Collinear gene pairs were defined based on at least five consecutive homologous gene pairs within syntenic blocks using an e-value threshold of 1 × 10−5. Gene duplication patterns were classified as follows [31]: (i) All genes were initially classified as ‘singletons’ and assigned gene ranks according to their order of appearance along chromosomes. (ii) Blastp results were evaluated, and the genes with blastp hits to other genes were re-labeled as ‘dispersed duplicates’. (iii) Genes with a rank difference of 1 were re-labeled as ‘tandem duplicates’. (iv) Genes with a rank difference of <20 were re-labeled as ‘proximal duplicates’. (v) The anchor genes in collinear blocks were re-labeled as ‘WGD/segmental’. All types of genomic transposable elements (TEs) were detected and annotated to explore their potential roles in shaping the NPC2 duplication pattern using the HiTE v3.1.2 software (95% length coverage threshold) [34]. Gene family sequences were compared with annotated TE sequences using MEGA software (version 11) [26], and transposon frequencies within the 5 kb upstream and downstream regions of duplicated and non-duplicated genes were calculated and visualized using ggplot2 v2.1.0 to investigate the potential roles of TEs in gene duplication [35].

2.6. An Analysis of the Cis-Regulatory Elements

The 2 kb-sequence upstream and downstream of the NPC2 start and end codon sites in the five Papilionidae species presented in this study were extracted and used for a cis-regulatory element analysis. Identification of cis-regulatory elements was performed using the plantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 November 2023) [36] to investigate the potential local adaptation mechanism through comparisons of Parnassius species with two other papilionids and through comparisons of P. glacialis with two other Parnassius species.

2.7. Genome Annotation and Kyoto Encyclopedia of Genes and Genome (KEGG) Pathway Enrichment

Gene annotation was performed using three methods: ab initio prediction, homology alignment, and RNA-seq support. Genescan v1.0 [37], Geneid v1.4 [38], SNAP v2013 [39], GlimmerHMM v3.04 [40], and Augustus v2.4 [41] tools were used for ab initio gene prediction. The homolog method was carried out using the software GeMoMa v1.3.1 [42] with default parameters. Functional annotations were obtained by searching the databases of NCBI-NR, Kyoto Encyclopedia of Genes and Genomes (KEGG) [43], Pfam [44], and SwissProt [45]. Gene functions for P. glacialis were assigned by aligning the protein sequences to the Kyoto Encyclopedia of Genes and Genome (KEGG) using Blastp (with the E-value threshold of ≤1 × 10−5), followed by a KEGG enrichment analysis of PgNPC2 genes using TBtools, with a Benjamini–Hochberg-adjusted p-value < 0.05 as the significance threshold [46,47].

2.8. RNA Extraction and Quantitative RT-PCR

About ten P. glacialis individuals (adults and fourth instar larvae) were collected at Mountain Laoshan, Nanjing, China. After sample collection, the fresh samples were stored at −80 °C until RNA extraction. The total RNA from different tissues of larval (head and gonad) and adult (head, antennae, thorax muscle, and leg) samples were extracted using Trizol Reagent (Vazyme, Nanjing, China), and reversed cDNA was synthesized using the PrimeScriptTM 1st stand cDNA Synthesis Kit (Takara, Shanghai, China). The primers (Table S1) for PgNPC2 gene amplification were synthesized by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). The PCR reaction was performed using the following procedure: 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The gene expression levels were analyzed using the comparative CT method (2−∆∆CT method) with β-actin gene as the reference [48].

3. Results

3.1. Genome-Wide Identification and Physicochemical Properties of NPC2 Genes

In this study, a total of 30 NPC2 genes were identified from chromosome-level genomes of 11 representative butterfly species (Table 1). Remarkably, ten NPC2 genes were identified in P. glacialis, which were sequentially renamed from the PgNPC2a to the PgNPC2j according to their chromosomal locations, while only one or two NPC2 genes were found in the other ten butterfly species. The sequence and physicochemical properties of these NPC2 genes, including the protein length (from 103 aa to 192 aa), molecular weights (from 11.13 kDa to 21.71 kDa), theoretical isoelectric points (from 4.60 to 9.02), instability indices (from 27.47 to 58.35), lipolysis indices (from 76.67 to 114.78), and total mean hydrophilicity indices (from −0.280 to 0.526), are shown in Table 1. In light of these datasets, the amino acids, molecular weights, and instability indices of Parnassius were relatively larger than those of other butterfly species. Furthermore, all PgNPC2 genes were shown to be located in the cytoplasm (Table S2), each harboring a typical signal peptide on their N-terminal ends.

3.2. A Phylogenetic Analysis of the NPC2 Gene Family Members in Representative Butterfly Genomes

All 30 NPC2 sequences of 11 butterfly species were selected for a comparative analysis in this study to explore their evolutionary relationships (Figure S1). The reconstructed NJ tree showed that the NPC2 genes of the butterflies in this study were divided into three subgroups (Figure 1), namely Cluster I (11 gene members), Cluster II (10 gene members), and Cluster III (9 gene members). In Cluster I, PgNPC2i from P. glacialis, PoNPC2b from P. orleans, and PaNPC2c from P. apollo shared the most common ancestor with high support values, and this small grouping was also found with other butterfly NPC2 genes, while Cluster II contained PgNPC2j from P. glacialis, PoNPC2a from P. orleans, and PaNPC2a and PaNPC2b from P. apollo, which also shared a common ancestor with high support values. In contrast, Cluster III contained eight NPC2 genes from P. glacialis and only one NPC2 gene (PaNPC2d) from P. apollo. Thus, compared with other butterfly species that harbored only one or two NPC2 genes, P. glacialis might have undergone a rapid expansion of NPC2 genes.

3.3. Conserved Motifs, Characteristic Domains, and Gene Structures of NPC2 Genes

A comprehensive analysis of the NPC2 properties (Figure 2A and Figure S3) showed that in total, 14 conserved motifs (motifs 1–14) were identified among the 30 NPC2 gene members using the online MEME and TBtools-II software, with the lengths ranging from 6 to 50 bp (Figure 2A). Interestingly, all NPC2 genes contained motifs 1 and 3 (Figure 2B), which are likely key components involved in functional specificity, and they were found in Clusters I and II along with motif 4, and Cluster III contained motifs 1, 2, 3, and 8.
A conserved domain analysis showed that only one conserved domain (MD-2-related lipid-recognition, ML) existed in all 30 NPC2 members of the butterflies (Figure 2A). The intron–exon structure analysis showed that all NPC2 gene members of the same cluster exhibited similar structures, that is, they shared identical exon numbers (Figure 2A). Interestingly, PgNPC2j, a member of Clusters I and II, contains three exons and two exceptionally long introns, while PgNPC2i contains only one exon and lacks introns; however, the PgNPC2 genes from Cluster III all contain four exons and three introns (Figure 2A).

3.4. Chromosomal Location, Gene Duplication, and Collinearity Analysis

A chromosomal location analysis showed that the NPC2 genes of the Parnassius butterfly species were all located on one or two chromosomes, and for P. glacialis, its 10 PgNPC2 genes were unevenly located on two chromosomes: the majority of PgNPC2 genes (eight genes) were on the chromosome Hic-asm-2, while two PgNPC2 genes were on the chromosome Hic-asm-11 (Figure 3).
A gene duplication analysis showed that a total of five tandem duplication NPC2 gene pairs were located on chromosomes Hic-asm-2 (PgNPC2c/PgNPC2d, PgNPC2e/PgNPC2f, PgNPC2f/PgNPC2g, and PgNPC2g/PgNPC2h) and Hic-asm-11 (PgNPC2i/PgNPC2j) (Figure 3). Additionally, PgNPC2a and PgNPC2b on Hic-asm-2 are proximal duplicates, suggesting that a complex pattern of gene duplication events existed in this genomic region. A gene duplication mechanism analysis showed that six major types of transposable elements, including DNA/hAT, RC/Helitron, LINE/RTE, LINE/L1, LTR/Pao, and LTR/Gypsy, were responsible for these duplication events (Table 2 and Table S4), and furthermore, within these transposon-mediated genes, PgNPC2g, PgNPC2h, and PgNPC2j were fully and partially transposon mediated, while the other seven genes were partially transposon-derived sequences (Figure 3). For the Hic-asm-2 chromosome, the PgNPC2 genes all contained the RC/Helitron and DNA/hAT transposons, with the exception of PgNPC2a and PgNPC2g lacking DNA/hAT. Additionally, most PgNPC2 genes were 1000 bp upstream or downstream and linked with the LTR/Gypsy retrotransposons, while PgNPC2c, PgNPC2d, PgNPC2e, and PgNPC2j were all derived from the insertion of LINE/RTE transposons. To investigate the potential role of TEs in mediating gene duplication events, we compared the sequences of gene family members with annotated and identified TE sequences. We found that an RC/Helitron transposon present in all PgNPC2 genes on the chromosome Hic-asm-2 is highly similar to the RC/Helitron transposon in the PgNPC2j gene on the chromosome Hic-asm-11 (Figure S2). In addition, by calculating the frequencies of transposons within the 5 kb upstream and downstream regions of duplicated and non-duplicated genes, it was found that the frequency of transposons was significantly higher near the duplicated genes, suggesting that a potential association exists between TEs and gene duplication (Figure S3).
The collinear relationships between 10 PgNPC2 genes and 9 other papilionid NPC2 genes are shown in Figure 4. Gene pairs (PbNPC2b/PgNPC2j, PmNPC2b/PgNPC2j, PaNPC2a/PgNPC2j, and PoNPC2b/PgNPC2j) were located on the chromosomal locations Pb-HIC_scaffold_12, Pm-NW_014496633.1, Pa-CAJQZP010000288.1, Pg-HIC_asm_11, and Po-ctg18 of five species (Pa. bianor, Pa. machaon, P. apollo, P. glacialis, and P. orleans), respectively; another one (PaNPC2d/PgNPC2b) was located on the chromosomes Pa-CAJQZP010001449.1 and Pg-HIC_asm_2 of P. glacialis and P. apollo. Obviously, there were four unique tandem repeats of P. glacialis NPC2 genes (PgNPC2c/PgNPC2d, PgNPC2e/PgNPC2f, PgNPC2f/PgNPC2g and PgNPC2g/PgNPC2h) that were likely to be newly duplicated.

3.5. Analysis of Cis-Regulatory Elements of NPC2 Genes

Different cis-regulatory elements within the gene promoter can result in distinct gene expression patterns [49]. Our analysis of the cis-acting elements showed that all five butterfly species contained light-responsive cis-acting elements; the cis-acting elements related to anaerobic induction, low-temperature acclimatization, and stress responses were especially rich in the Parnassius genomes, and other cis-acting elements, such as the binding site of AT-rich DNA binding protein (ATBP-1) and a wound-responsive element, were detected in P. glacialis (Figure 5).

3.6. The KEGG Enrichment Analysis for PgNPC2 Genes of P. glacialis

The KEGG enrichment analysis results indicate that the PgNPC2 gene of P. glacialis was significantly enriched in pathways related to cholesterol metabolism, lysosomal metabolism, digestive system, transport and catabolism, cellular processes, organismal systems, and signaling and cellular pathways. Among these pathways, the cholesterol metabolism harbored the highest significance (−Log10(p-value) ~18), while the signaling and cellular pathways showed the lowest significance (−Log10(p-value) > 6) (Figure 6, Table S5).

3.7. The Variance of NPC2 Gene Expression Levels in Different Tissues of P. glacialis

All ten PgNPC2 genes in the P. glacialis genome were used for qRT-PCR verification, and the results indicate that all PgNPC2 genes were expressed at relatively low levels in the thoraxes, legs, and gonads of the adults, as well as in the heads and gonads of the larvae; however, nine of the ten PgNPC2 genes were highly expressed in the antennae and moderately expressed in the heads of the adults. These facts indicate that the adult P. glacialis antennae and heads were likely to play a key role in the local adaptation of olfactory communication, host plant recognition, and reproduction through high-level PgNPC2 expression (Figure 7 and Table S4).

4. Discussion

In this study, a total of 10 genes of the NPC2 gene family in P. glacialis were identified for the first time through a genome-wide analysis of the butterflies available. All PgNPC2 genes contain a highly conserved MD-2-related lipid-recognition (ML) domain and six cysteines, which is consistent with other insect groups [15,19]. However, our analysis revealed that, compared to other butterfly species, the P. glacialis had a significant expansion of NPC2 genes, which could have been driven by tandem duplications mediated by transposon insertions, causing motif structure changes accompanied by intron gains or losses [50].
Previous studies showed that as the distributional elevation increased, the genome size of the Parnassius butterflies generally decreased, as in case of the three Parnassius species (P. orleans (1.23 Gb), P. apollo (~1.39), and P. glacialis (~1.35 Gb)) in this study. Our phylogenetic and collinearity analyses revealed that the Parnassius species, especially P. apollo and P. glacialis, harbored relatively larger amounts of NPC2 genes (four and ten, respectively) than other butterfly groups (one or two). Phylogenetically, the eight PgNPC2 genes and PaNPC2d in Cluster III shared the nearest common ancestor, with the former gene clusters being shaped by rapid tandem duplications [4,8]. These expanded genes in Parnassius may have contributed to variations in lipid or cholesterol metabolism (Figure 6), potentially providing a selective advantage at the relatively low-altitude habitats in which the oxygen content and temperature are relatively higher; in addition, the NPC2 expansions might also be somewhat attributed to the genome size increases that provided genetic make-ups for local adaptation to distinct ecological niches [8,32].
Gene duplication was shown to be one of the key factors in shaping genome diversity for adaptive evolution [48,51,52,53,54,55,56]. For the expansion of gene family, tandem repeat and TE-mediated duplication are the two main mechanisms [55,56]. A large amount of PgNPC2 tandem duplications in P. glacialis were also found in this study, verifying the fact that the tandem repeat of functional genes is probably associated with local adaptation, as shown in previous studies. For example, P450 tandem duplications were shown to be linked to the biosynthesis of defense compounds, pigments, antioxidants, and detoxification processes, as well as to adaptation to environmental changes, in coffee, tomato, and other Solanaceae plants [55,57]. Recently, TEs, especially LTR-TEs were identified as the major drivers of genome evolutionary adaptation [58,59], and other TEs, such as the Helitron, were also shown to probably facilitate gene duplication in maize and bats by capturing roll-loop-mediated gene fragments [60,61]. Our study revealed that the P. glacialis Helitron transposons were responsible for the duplication of PgNPC2 genes on the Hic-asm-2 chromosome, while the LINE and LTR retrotransposons likely contributed to intronless pseudogenes, a few of which, such as the PgNPC2i, showed a detectable level of expressions through qRT-PCR (Figure 7), as found in previous studies [62]; however, further functional assays are required to confirm their physiological roles.
Our cis-acting element analysis showed that the five papilionid butterfly species all contained light-responsive cis-acting elements that were associated with circadian rhythms and behaviors of forage and migration [63]; in addition, the Parnassius species was especially enriched with elements related to anaerobic induction, low-temperature acclimatization, and environmental stress responses, which were likely to be responsible for their adaptation to high-altitude environments. Furthermore, the ATBP-1 binding sites and wound-responsive elements found only in P. glacialis might be involved in wound response and immune defense to pathogen invasion and stress in relatively low-altitude mountain areas [64].
Additionally, our qRT-PCR analysis revealed that a total of nine NPC2 genes were highly expressed in the head, especially in the antennae; however, these NPC2 genes were lowly expressed in other tissues of the P. glacialis. As far as we know, NPC2 genes are mainly involved in olfactory communication, which influence the habitat selection and mate recognition of insects [15,17,19], and thus, the P. glacialis antennae and other sensory organs on the head might play a potential role in the species’ ‘out of Qinghai-Tibet Plateau’ dispersal from a northeastern direction. However, further functional tests are also needed to confirm the specific roles of these genes in olfactory communication and dispersal adaptation.

5. Conclusions

The study analyzed the NPC2 gene family in the genomes of 11 representative butterfly species, including 3 Parnassius species, and systematically examined the gene structures and phylogenetic relationships with a focus on the gene duplication and expression patterns of P. glacialis. The results indicate that the NPC2 genes of P. glacialis had a significant expansion driven by tandem duplications mediated by transposon insertions. Furthermore, the qRT-PCR analysis showed that NPC2 genes were highly expressed in the antenna, followed by the head, with low expression in other tissues. This study provides a comprehensive overview of the NPC2 gene family of P. glacialis and offers new insights into the local adaptation mechanisms of this alpine butterfly species.

Supplementary Materials

The following supporting information can be downloaded at www.mdpi.com/article/10.3390/genes16030249/s1, Figure S1: Alignment of amino acid sequences of NPC2 proteins from 11 butterfly species; Box represents conserved domain of NPC2 gene family, with black and gray shaded areas representing conserved sites; Figure S2: Transposon sequence alignment in the PgNPC2 genes of P. glacialis. The black and gray areas represent highly conserved transposon sites in different PgNPC2 genes; Figure S3: Transposon frequencies in the upstream and downstream 5 kb regions of duplicated genes and non-duplicated genes of the NPC2 genes in the genome of P. glacialis. The vertical axis represents the frequency of transposon occurrence, with the red box plot representing the downstream region of genes and the blue box plot representing the upstream region of genes; Table S1: Primer information of the PgNPC2 genes; Table S2: Secondary structure and subcellular localization of PgNPC2 genes of P. glacialis; Table S3: Information of identified motifs; Table S4: Transposable element information in PgNPC2 genes of P. glacialis; Table S5: Information about KEGG pathways for PgNPC2 genes of P. glacialis; Table S6: Sampling information about P. glacialis for qRT-PCR.

Author Contributions

Conceptualization, Z.Z., C.S. and X.G.; Methodology, Z.Z. and C.S.; Software, C.S. and Y.Z.; Validation, Z.Z., C.S., Y.Z., R.N. and B.H.; Formal Analysis, Z.Z., C.S. and Y.Z.; Investigation, R.N. and B.H.; Resources, Z.Z.; Data Curation, Z.Z.; Writing—Original Draft Preparation, Z.Z. and C.S.; Writing—Review and Editing, J.H.; funding acquisition, J.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Science Foundation of China (No.41972029) and the Natural Science Foundation of Universities of Anhui Province (No. KJ2021A100).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome data of P. glacialis were deposited into GenBank with the BioProject number PRJNA893814.

Acknowledgments

We thank all other members of our laboratory for their useful suggestions and support. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Xu, W.; Dong, W.J.; Fu, T.T.; Gao, W.; Lu, C.Q.; Yan, F.; Wu, Y.H.; Jiang, K.; Jin, J.Q.; Chen, H.M.; et al. Herpetological phylogeographic analyses support a Miocene focal point of Himalayan uplift and biological diversification. Natl. Sci. Rev. 2021, 8, nwaa263. [Google Scholar] [CrossRef] [PubMed]
  2. Condamine, F.L.; Rolland, J.; Höhna, S.; Sperling, F.A.H.; Sanmartín, I. Testing the role of the Red Queen and Court Jester as drivers of the macroevolution of Apollo butterflies. Natl. Sci. Rev. 2018, 67, 940–964. [Google Scholar] [CrossRef] [PubMed]
  3. Zhao, D.N.; Ren, Y.; Zhang, J.Q. Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau. Natl. Sci. Rev. 2020, 144, 106713. [Google Scholar] [CrossRef]
  4. Su, C.Y.; Ding, C.; Zhao, Y.J.; He, B.; Nie, R.E.; Hao, J.S. Diapause-Linked gene expression pattern and related candidate duplicated genes of the mountain butterfly Parnassius glacialis (Lepidoptera: Papilionidae) revealed by comprehensive transcriptome profiling. Int. J. Mol. Sci. 2023, 24, 5577. [Google Scholar] [CrossRef] [PubMed]
  5. He, B.; Zhao, Y.J.; Su, C.Y.; Lin, G.H.; Wang, Y.L.; Li, L.; Ma, J.Y.; Yang, Q.; Hao, J.S. Phylogenomics reveal extensive phylogenetic discordance due to incomplete lineage sorting following the rapid radiation of alpine butterflies (Papilionidae: Parnassius). Syst. Entomol. 2023, 48, 585–599. [Google Scholar] [CrossRef]
  6. Galetti, M.; Moleon, M.; Jordano, P.; Pires, M.M.; Guimaraes, P.R., Jr.; Pape, T.; Nichols, E.; Hansen, D.; Olesen, J.M.; Munk, M.; et al. Ecological and evolutionary legacy of megafauna extinctions. Biol. Rev. 2018, 93, 845–862. [Google Scholar] [CrossRef] [PubMed]
  7. Tao, R.S.; Xu, C.; Wang, Y.L.; Sun, X.Y.; Li, C.X.; Ma, J.Y.; Hao, J.S.; Yang, Q. Spatiotemporal differentiation of alpine butterfly Parnassius glacialis (Papilionidae: Parnassiinae) in China: Evidence from mitochondrial DNA and nuclear single nucleotide polymorphisms. Genes 2020, 11, 188. [Google Scholar] [CrossRef] [PubMed]
  8. Zhao, Y.J.; Su, C.Y.; He, B.; Nie, R.E.; Wang, Y.; Ma, J.Y.; Song, J.Y.; Yang, Q.; Hao, J.S. Dispersal from the Qinghai-Tibet plateau by a high-altitude butterfly is associated with rapid expansion and reorganization of its genome. Nat. Commun. 2023, 14, 8190. [Google Scholar] [CrossRef]
  9. Ding, C.; Su, C.Y.; Li, Y.L.; Zhao, Y.J.; Wang, Y.L.; Wang, Y.; Nie, R.E.; He, B.; Ma, J.Y.; Hao, J.S. Interspecific and intraspecific transcriptomic variations unveil the potential high-altitude adaptation mechanisms of the Parnassius butterfly species. Genes 2024, 15, 1013. [Google Scholar] [CrossRef] [PubMed]
  10. Pelosi, P.; Iovinella, I.; Felicioli, A.; Dani, F.R. Soluble proteins of chemical communication: An overview across arthropods. Front. Physiol. 2014, 5, 320. [Google Scholar] [CrossRef] [PubMed]
  11. Storch, J.; Xu, Z. Niemann-Pick C2 (NPC2) and intracellular cholesterol trafficking. Biochim. Biophys. Acta 2009, 1791, 671–678. [Google Scholar] [CrossRef]
  12. Qian, H.W.; Wu, X.L.; Du, X.M.; Yao, X.; Zhao, X.; Lee, J.C.; Yang, H.Y.; Yan, N. Structural basis of Low-pH-Dependent lysosomal cholesterol egress by NPC1 and NPC2. Cell 2020, 182, 98–111.e18. [Google Scholar] [CrossRef]
  13. Xu, Y.; Zhang, Q.; Tan, L.; Xie, X.; Zhao, Y. The characteristics and biological significance of NPC2: Mutation and disease. Mutat. Res. Rev. Mutat. Res. 2019, 782, 108284. [Google Scholar] [CrossRef]
  14. Sleat, D.E.; Wiseman, J.A.; El-Banna, M.; Price, S.M.; Verot, L.; Shen, M.M.; Tint, G.S.; Vanier, M.T.; Walkley, S.U.; Lobel, P. Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc. Natl. Acad. Sci. USA 2004, 101, 5886–5891. [Google Scholar] [CrossRef]
  15. Zhang, L.Z.N.; Jiang, H.; Wu, F.; Li, H. Molecular cloning and expression pattern analysis of NPC2 gene family of Apis cerana cerana. Sci. Agric. Sin. 2022, 55, 2461–2471. [Google Scholar]
  16. Huang, X.; Warren, J.T.; Buchanan, J.; Gilbert, L.I.; Scott, M.P. Drosophila Niemann-Pick type C-2 genes control sterol homeostasis and steroid biosynthesis: A model of human neurodegenerative disease. Development 2007, 134, 3733–3742. [Google Scholar] [CrossRef] [PubMed]
  17. Ishida, Y.; Tsuchiya, W.; Fujii, T.; Fujimoto, Z.; Miyazawa, M.; Ishibashi, J.; Matsuyama, S.; Ishikawa, Y.; Yamazaki, T. Niemann-Pick type C2 protein mediating chemical communication in the worker ant. Proc. Natl. Acad. Sci. USA 2014, 111, 3847–3852. [Google Scholar] [CrossRef] [PubMed]
  18. Shi, X.Z.; Zhong, X.; Yu, X.Q. Drosophila melanogaster NPC2 proteins bind bacterial cell wall components and may function in immune signal pathways. Insect Biochem. Mol. Biol. 2012, 42, 545–556. [Google Scholar] [CrossRef]
  19. Zheng, Y.; Wang, S.N.; Peng, Y.; Lu, Z.Y.; Shan, S.; Yang, Y.Q.; Li, R.J.; Zhang, Y.J.; Guo, Y.Y. Functional characterization of a Niemann-Pick type C2 protein in the parasitoid wasp Microplitis mediator. Insect. Sci. 2018, 25, 765–777. [Google Scholar] [CrossRef] [PubMed]
  20. Podsiadlowski, L.; Tunstrom, K.; Espeland, M.; Wheat, C.W. The genome assembly and annotation of the Apollo butterfly Parnassius apollo, a flagship species for conservation biology. Genome Biol. Evol. 2021, 13, evab122. [Google Scholar] [CrossRef] [PubMed]
  21. He, J.W.; Zhang, R.; Yang, J.; Chang, Z.; Zhu, L.X.; Lu, S.H.; Xie, F.A.; Mao, J.L.; Dong, Z.W.; Liu, G.C.; et al. High-quality reference genomes of swallowtail butterflies provide insights into their coloration evolution. Zool. Res. 2022, 43, 367–379. [Google Scholar] [CrossRef]
  22. Lu, S.; Yang, J.; Dai, X.; Xie, F.; He, J.; Dong, Z.; Mao, J.; Liu, G.; Chang, Z.; Zhao, R.; et al. Chromosomal-level reference genome of Chinese peacock butterfly (Papilio bianor) based on third-generation DNA sequencing and Hi-C analysis. GigaScience 2019, 8, giz128. [Google Scholar] [CrossRef] [PubMed]
  23. Lewis, J.J.; van der Burg, K.R.L.; Mazo-Vargas, A.; Reed, R.D. ChIP-Seq-Annotated Heliconius erato genome highlights patterns of cis-regulatory evolution in Lepidoptera. Cell Rep. 2016, 16, 2855–2863. [Google Scholar] [CrossRef] [PubMed]
  24. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
  25. Horton, P.; Park, K.J.; Obayashi, T.; Fujita, N.; Harada, H.; Adams-Collier, C.J.; Nakai, K. WoLF PSORT: Protein localization predictor. Nucleic Acids Res. 2007, 35, W585–W587. [Google Scholar] [CrossRef] [PubMed]
  26. Tamura, K.; Stecher, G.; Kumar, S.; Battistuzzi, F.U. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  27. Capella-Gutiérrez, S.; Silla-Martínez, J.M.; Gabaldón, T. TrimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009, 25, 1972–1973. [Google Scholar] [CrossRef] [PubMed]
  28. Letunic, I.; Bork, P. Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 2024, 52, W78–W82. [Google Scholar] [CrossRef]
  29. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  30. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef]
  31. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
  32. Qi, T.S.; He, F.M.; Zhang, X.Q.; Wang, J.Q.; Zhang, Z.L.; Jiang, H.R.; Zhao, B.A.; Du, C.; Che, Y.Z.; Feng, X.; et al. Genome-wide identification and expression profiling of Potato (Solanum tuberosum L.) universal stress proteins reveal essential roles in mechanical damage and deoxynivalenol stress. Int. J. Mol. Sci. 2024, 25, 1341. [Google Scholar] [CrossRef] [PubMed]
  33. Lv, X.M.; Tian, S.C.; Huang, S.L.; Wei, G.B.; Han, D.M.; Li, J.G.; Guo, D.L.; Zhou, Y. Genome-wide identification of the longan R2R3-MYB gene family and its role in primary and lateral root. BMC Plant Biol. 2023, 23, 448. [Google Scholar] [CrossRef]
  34. Hu, K.; Ni, P.; Xu, M.H.; Zou, Y.; Chang, J.Y.; Gao, X.; Li, Y.H.; Ruan, J.; Hu, B.; Wang, J.X. HiTE: A fast and accurate dynamic boundary adjustment approach for full-length transposable element detection and annotation. Nat. Commun. 2024, 15, 5573. [Google Scholar] [CrossRef]
  35. Ginestet, C. ggplot2: Elegant graphics for data analysis. J. R. Stat. Soc. Ser. A Stat. Soc. 2011, 174, 245–246. [Google Scholar] [CrossRef]
  36. Lescot, M.; Dehais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouze, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  37. Burge, C.; Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 1997, 268, 78–94. [Google Scholar] [CrossRef] [PubMed]
  38. Parra, G.; Blanco, E.; Guigó, R. GeneID in Drosophila. Genome Res. 2000, 10, 511–515. [Google Scholar] [CrossRef]
  39. Birney, E.; Clamp, M.; Durbin, R. GeneWise and genomewise. Genome Res. 2004, 14, 988–995. [Google Scholar] [CrossRef]
  40. Majoros, W.H.; Pertea, M.; Salzberg, S.L. TigrScan and GlimmerHMM: Two open source ab initio eukaryotic gene-finders. Bioinformatics 2004, 20, 2878–2879. [Google Scholar] [CrossRef] [PubMed]
  41. Stanke, M.; Keller, O.; Gunduz, I.; Hayes, A.; Waack, S.; Morgenstern, B. AUGUSTUS: A web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 2005, 33, W465–W467. [Google Scholar] [CrossRef]
  42. Keilwagen, J.; Hartung, F.; Grau, J. GeMoMa: Homology-based gene prediction utilizing intron position conservation and RNA-seq data. Methods Mol. Biol. 2019, 1962, 161–177. [Google Scholar]
  43. Kanehisa, M.; Sato, Y.; Kawashima, M.; Furumichi, M.; Ishiguro-Watanabe, M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016, 44, D457–D462. [Google Scholar] [CrossRef] [PubMed]
  44. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef] [PubMed]
  45. Bateman, A. UniProt: The universal protein knowledgebase in 2021. Nucleic Acids Res. 2021, 49, D480–D489. [Google Scholar]
  46. Zhang, Y.H.; Chu, C.; Wang, S.; Chen, L.; Lu, J.; Kong, X.; Huang, T.; Li, H.; Cai, Y.D. The use of Gene Ontology term and KEGG pathway enrichment for analysis of drug half-life. PLoS ONE 2016, 11, e0165496. [Google Scholar] [CrossRef] [PubMed]
  47. Kanehisa, M.; Furumichi, M.; Sato, Y.; Kawashima, M.; Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023, 51, D587–D592. [Google Scholar] [CrossRef] [PubMed]
  48. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  49. Oudelaar, A.M.; Higgs, D.R. The relationship between genome structure and function. Nat. Rev. Genet. 2021, 22, 154–168. [Google Scholar] [CrossRef]
  50. Fan, K.; Shen, H.; Bibi, N.; Li, F.; Yuan, S.; Wang, M.; Wang, X. Molecular evolution and species-specific expansion of the NAP members in plants. J. Integr. Plant. Biol. 2015, 57, 673–687. [Google Scholar] [CrossRef] [PubMed]
  51. Long, M.; Thornton, K. Gene duplication and evolution. Science 2001, 293, 1551. [Google Scholar] [CrossRef] [PubMed]
  52. Demuth, J.P.; De Bie, T.; Stajich, J.E.; Cristianini, N.; Hahn, M.W. The evolution of mammalian gene families. PLoS ONE 2006, 1, e85. [Google Scholar] [CrossRef] [PubMed]
  53. Flagel, L.E.; Wendel, J.F. Gene duplication and evolutionary novelty in plants. New Phytol. 2009, 183, 557–564. [Google Scholar] [CrossRef] [PubMed]
  54. Canestro, C.; Albalat, R.; Irimia, M.; Garcia-Fernandez, J. Impact of gene gains, losses and duplication modes on the origin and diversification of vertebrates. Cell Dev. Biol. 2013, 24, 83–94. [Google Scholar] [CrossRef]
  55. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef] [PubMed]
  56. Tan, S.; Ma, H.; Wang, J.; Wang, M.; Wang, M.; Yin, H.; Zhang, Y.; Zhang, X.; Shen, J.; Wang, D.; et al. DNA transposons mediate duplications via transposition-independent and dependent mechanisms in metazoans. Nat. Commun. 2021, 12, 4280. [Google Scholar] [CrossRef]
  57. Yu, J.; Tehrim, S.; Wang, L.; Dossa, K.; Zhang, X.; Ke, T.; Liao, B. Evolutionary history and functional divergence of the cytochrome P450 gene superfamily between Arabidopsis thaliana and Brassica species uncover effects of whole genome and tandem duplications. BMC Genom. 2017, 18, 733. [Google Scholar] [CrossRef]
  58. Gonzalez, J.; Lenkov, K.; Lipatov, M.; Macpherson, J.M.; Petrov, D.A. High rate of recent transposable element-induced adaptation in Drosophila melanogaster. PLoS Biol. 2008, 6, e251. [Google Scholar] [CrossRef] [PubMed]
  59. Oliver, K.R.; Greene, W.K. Transposable elements: Powerful facilitators of evolution. Bioessays 2009, 31, 703–714. [Google Scholar] [CrossRef] [PubMed]
  60. Morgante, M.; Brunner, S.; Pea, G.; Fengler, K.; Zuccolo, A.; Rafalski, A. Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize. Nat. Genet. 2005, 37, 997–1002. [Google Scholar] [CrossRef]
  61. Grabundzija, I.; Messing, S.A.; Thomas, J.; Cosby, R.L.; Bilic, I.; Miskey, C.; Gogol-Doring, A.; Kapitonov, V.; Diem, T.; Dalda, A.; et al. A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes. Nat. Commun. 2016, 7, 10716. [Google Scholar] [CrossRef]
  62. Casola, C.; Betran, E. The genomic impact of gene retrocopies: What have we learned from comparative genomics, population genomics, and transcriptomic analyses? Genome Biol. Evol. 2017, 9, 1351–1373. [Google Scholar] [CrossRef] [PubMed]
  63. Saunders, D.S. Dormancy, diapause, and the role of the circadian system insect photoperiodism. Annu. Rev. Entomol. 2020, 65, 373–389. [Google Scholar] [CrossRef] [PubMed]
  64. Srivastava, S.; Pandey, S.P.; Singh, P.; Pradhan, L.; Pande, V.; Sane, A.P. Early wound-responsive cues regulate the expression of WRKY family genes in chickpea differently under wounded and unwounded conditions. Physiol. Mol. Biol. Plants 2022, 28, 719–735. [Google Scholar] [CrossRef]
Genes 16 00249 g001
Figure 1. The phylogenetic relationships of the NPC2 genes of the 11 representative butterfly species. The phylogenetic tree was reconstructed with the neighbor-joining (NJ) method through 1000 bootstrap replicates using MEGA 11.0.13. Different arcs indicate different subgroups (Cluster I–Cluster III). Different colors represent NPC2 genes of different butterfly species.
Figure 1. The phylogenetic relationships of the NPC2 genes of the 11 representative butterfly species. The phylogenetic tree was reconstructed with the neighbor-joining (NJ) method through 1000 bootstrap replicates using MEGA 11.0.13. Different arcs indicate different subgroups (Cluster I–Cluster III). Different colors represent NPC2 genes of different butterfly species.
Genes 16 00249 g001
Genes 16 00249 g002
Figure 2. Structural characteristics of NPC2 genes based on their phylogeny. (A) Left, schematic representation of conserved motifs of 30 NPC2 proteins with different colored rectangles representing conserved motifs 1–14. (A) Middle, conserved domains, with green boxes representing MD-2-related lipid-recognition (ML) domain and gray boxes not containing domains. (A) Right, structure of exons (pink boxes) and introns (black lines) of NPC2 genes, with green rectangles representing 3′ or 5′ end non-coding regions. (B) Conserved motifs: motif 1 and motif 3.
Figure 2. Structural characteristics of NPC2 genes based on their phylogeny. (A) Left, schematic representation of conserved motifs of 30 NPC2 proteins with different colored rectangles representing conserved motifs 1–14. (A) Middle, conserved domains, with green boxes representing MD-2-related lipid-recognition (ML) domain and gray boxes not containing domains. (A) Right, structure of exons (pink boxes) and introns (black lines) of NPC2 genes, with green rectangles representing 3′ or 5′ end non-coding regions. (B) Conserved motifs: motif 1 and motif 3.
Genes 16 00249 g002
Genes 16 00249 g003
Figure 3. Chromosomal locations of the P. orleans, P. apollo, and P. glacialis NPC2 genes. (A) P. orlens chromosomal contig18. (B,C) P. apollo chromosomal contigCAJQZP010000288.1 and contigCAJQZP010001449.1. (D,E) P. glacialis chromosomes Hic-asm-2 and Hic-asm-11. Tandem duplicated gene pairs of P. glacialis are shown in the red arc. The green boxes represent complete transposons, the blue boxes represent partial transposons, and the yellow boxes represent LTR/Gypsy transposons.
Figure 3. Chromosomal locations of the P. orleans, P. apollo, and P. glacialis NPC2 genes. (A) P. orlens chromosomal contig18. (B,C) P. apollo chromosomal contigCAJQZP010000288.1 and contigCAJQZP010001449.1. (D,E) P. glacialis chromosomes Hic-asm-2 and Hic-asm-11. Tandem duplicated gene pairs of P. glacialis are shown in the red arc. The green boxes represent complete transposons, the blue boxes represent partial transposons, and the yellow boxes represent LTR/Gypsy transposons.
Genes 16 00249 g003
Genes 16 00249 g004
Figure 4. A plot of interspecific collinearity among P. glacialis, Pa. bianor, Pa. machaon, P. apollo, P. glacialis, and P. orleans. The gene pairs are highlighted by the red lines, with duplicated genes shown nearby. The gray lines indicate collinear blocks between chromosomes or scaffolds of different species.
Figure 4. A plot of interspecific collinearity among P. glacialis, Pa. bianor, Pa. machaon, P. apollo, P. glacialis, and P. orleans. The gene pairs are highlighted by the red lines, with duplicated genes shown nearby. The gray lines indicate collinear blocks between chromosomes or scaffolds of different species.
Genes 16 00249 g004
Genes 16 00249 g005
Figure 5. Cis-regulatory elements (CREs) of the papilionid NPC2 genes. The black lines represent a 2 kb sequence upstream and downstream of the start and stop codon sites of NPC2 genes, and the different colored boxes correspond to different CREs.
Figure 5. Cis-regulatory elements (CREs) of the papilionid NPC2 genes. The black lines represent a 2 kb sequence upstream and downstream of the start and stop codon sites of NPC2 genes, and the different colored boxes correspond to different CREs.
Genes 16 00249 g005
Genes 16 00249 g006
Figure 6. Enriched KEGG pathways for the PgNPC2 genes in the P. glacialis genome. Each bar represents a different biological pathway. The x-axis represents the Benjamini–Hochberg-adjusted p-value, where the smaller the p-value, the higher the enrichment of the pathway. The y-axis represents the pathways identified in the KEGG enrichment analysis.
Figure 6. Enriched KEGG pathways for the PgNPC2 genes in the P. glacialis genome. Each bar represents a different biological pathway. The x-axis represents the Benjamini–Hochberg-adjusted p-value, where the smaller the p-value, the higher the enrichment of the pathway. The y-axis represents the pathways identified in the KEGG enrichment analysis.
Genes 16 00249 g006
Genes 16 00249 g007
Figure 7. The expression levels of the nine PgNPC2 genes in different P. glacialis tissues. The x-axis represents different tissues, and the y-axis represents the gene expression levels (relative expression, 2−ΔΔCt). The y-axis is truncated to better visualize differences in expression levels. The bar graph is enhanced with error bars to facilitate a more intuitive comparison of expression differences, and the different colors of the bars represent different NPC2 genes.
Figure 7. The expression levels of the nine PgNPC2 genes in different P. glacialis tissues. The x-axis represents different tissues, and the y-axis represents the gene expression levels (relative expression, 2−ΔΔCt). The y-axis is truncated to better visualize differences in expression levels. The bar graph is enhanced with error bars to facilitate a more intuitive comparison of expression differences, and the different colors of the bars represent different NPC2 genes.
Genes 16 00249 g007
Table 1. Physicochemical properties of NPC2 genes identified in this study.
Table 1. Physicochemical properties of NPC2 genes identified in this study.
SpeciesGene NameGene IDAmino Acid Number/aaMolecular Weight
/Da		Theoretical IsoelectricInstabilityStabilityLipolysis IndicesTotal Mean Hydrophilic Value
P. apolloPaNPC2aPapo004992.115417,122.95.7350.96unstable99.29−0.023
PaNPC2bPapo004994.111012,421.35.3853.96unstable87.64−0.093
PaNPC2cPapo004995.116718,299.38.6338.97stable93.890.116
PaNPC2dPapo023738.117018,628.98.9737.31stable98.120.094
P. glacialisPgNPC2aevm.model.CTG_193.5017419,187.24.649.94unstable98.450.326
PgNPC2bevm.model.CTG_193.6817018,719.68.2840.11unstable92.290.039
PgNPC2cevm.model.CTG_193.7017018,671.68.6340.36unstable94.590.066
PgNPC2devm.model.CTG_193.7117018,699.68.2839.11stable94.590.05
PgNPC2eevm.model.CTG_54.22316918,632.58.2842.1unstable92.840.027
PgNPC2fevm.model.CTG_54.22217018,699.68.2839.11stable94.590.05
PgNPC2gevm.model.CTG_54.22117018,607.48.2748.95unstable86−0.002
PgNPC2hevm.model.CTG_54.22017519,299.39.0241.31unstable89.09−0.11
PgNPC2ievm.model.CTG_162.12111412,533.55.1758.35unstable105.960.32
PgNPC2jevm.model.CTG_162.12015416,928.65.6351.2unstable94.87−0.008
P. orleansPoNPC2aevm.model.ctg18.15015416,968.66.0739.09stable92.34−0.103
PoNPC2bevm.model.ctg18.15110311,132.85.4556.63unstable97.380.256
Pi. napiPnNPC2aPnap002095.117519,309.58.0342.62unstable86.17−0.065
PnNPC2bPnap002017.116518,1058.3937.3stable81.52−0.108
Pi. rapaePrNPC2aPrap002064.116518,1297.542.05unstable86.24−0.127
PrNPC2bPrap002333.115617,0088.0727.47stable99.230.053
Pa. machaonPmNPC2aPmac012693.116618,221.16.7233.02stable94.40.051
Pa. bianorPbNPC2aPbia004755.112113,646.98.5845.37unstable82.07−0.086
PbNPC2bPbia004754.116618,2116.7133.5stable91.510.017
F. adippeFaNPC2aFadi023545.116518,367.38.2135.33stable92.67−0.096
FaNPC2bFadi023544.119021,707.58.950.38unstable89.11−0.056
H. eratoHeNPC2aHera002910.115717,543.65.2638.23stable114.780.526
H. commaHcNPC2aHcom020357.119221,082.28.838.06stable76.67−0.084
HcNPC2bHcom020358.113314,6647.6238.45stable82.03−0.095
C. argiolusCaNPC2aCarg014702.115617,534.28.4838.82stable85.51−0.28
CaNPC2bCarg004219.116317,910.66.4129.3stable89.02−0.064
Table 2. Major types of transposable elements in PgNPC2 genes of P. glacialis.
Table 2. Major types of transposable elements in PgNPC2 genes of P. glacialis.
Gene IDChromosomePosition RelationStartEndTransposon Type
PgNPC2aHic_asm_2gene contains TE13353301335554RC/Helitron
PgNPC2aHic_asm_2gene close to TE13383111338500LTR/Gypsy
PgNPC2bHic_asm_2gene contains TE25178252518233RC/Helitron
PgNPC2bHic_asm_2gene contains TE25195292519773LINE/RTE
PgNPC2bHic_asm_2gene close to TE25206252520760LTR/Gypsy
PgNPC2cHic_asm_2gene contains TE25922012592612RC/Helitron
PgNPC2cHic_asm_2gene contains TE25931772593421DNA/hAT
PgNPC2cHic_asm_2gene contains TE25937552594059LINE/RTE
PgNPC2cHic_asm_2gene close to TE25944902594684LTR/Gypsy
PgNPC2dHic_asm_2gene contains TE26092122609717RC/Helitron
PgNPC2dHic_asm_2gene contains TE26102072610263DNA/hAT
PgNPC2dHic_asm_2gene contains TE26120342612351LINE/RTE
PgNPC2eHic_asm_2gene contains TE26601982660703RC/Helitron
PgNPC2eHic_asm_2gene contains TE26609102661154DNA/hAT
PgNPC2eHic_asm_2gene contains TE26614892661741LINE/RTE
PgNPC2eHic_asm_2gene close to TE26622012662395LTR/Gypsy
PgNPC2fHic_asm_2gene contains TE27014892701971RC/Helitron
PgNPC2fHic_asm_2gene contains TE27022012702445DNA/hAT
PgNPC2fHic_asm_2gene contains TE27043932704618LINE/RTE
PgNPC2gHic_asm_2gene contains TE27121712712725RC/Helitron
PgNPC2gHic_asm_2gene contains TE27128452713124LINE/L1
PgNPC2hHic_asm_2gene contains TE27221182722514LTR/Gypsy
PgNPC2hHic_asm_2gene contains TE27231632723709RC/Helitron
PgNPC2hHic_asm_2gene contains TE27292182729589LTR/ERV
PgNPC2hHic_asm_2gene contains TE27297692730136DNA/hAT
PgNPC2iHic_asm_11gene overlaps TE1785701817857217DNA/PIF-Harbinger
PgNPC2iHic_asm_11gene close to TE1785842517858553LTR/Gypsy
PgNPC2jHic_asm_11gene contains TE1787514817878336LINE/RTE
PgNPC2jHic_asm_11gene contains TE1786667417867025DNA/TcMar
PgNPC2jHic_asm_11gene contains TE1787106517871268RC/Helitron
PgNPC2jHic_asm_11gene contains TE1787343317875156RC/Helitron
PgNPC2jHic_asm_11gene contains TE1787990717880988DNA/hAT
PgNPC2jHic_asm_11gene contains TE1789110817893662DNA/P
PgNPC2jHic_asm_11gene contains TE1789424617894618DNA/TcMar
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhu, Z.; Su, C.; Guo, X.; Zhao, Y.; Nie, R.; He, B.; Hao, J. Genome-Wide Identification, Gene Duplication, and Expression Pattern of NPC2 Gene Family in Parnassius glacialis. Genes 2025, 16, 249. https://doi.org/10.3390/genes16030249

AMA Style

Zhu Z, Su C, Guo X, Zhao Y, Nie R, He B, Hao J. Genome-Wide Identification, Gene Duplication, and Expression Pattern of NPC2 Gene Family in Parnassius glacialis. Genes. 2025; 16(3):249. https://doi.org/10.3390/genes16030249

Chicago/Turabian Style

Zhu, Zhenyao, Chengyong Su, Xuejie Guo, Youjie Zhao, Ruie Nie, Bo He, and Jiasheng Hao. 2025. "Genome-Wide Identification, Gene Duplication, and Expression Pattern of NPC2 Gene Family in Parnassius glacialis" Genes 16, no. 3: 249. https://doi.org/10.3390/genes16030249

APA Style

Zhu, Z., Su, C., Guo, X., Zhao, Y., Nie, R., He, B., & Hao, J. (2025). Genome-Wide Identification, Gene Duplication, and Expression Pattern of NPC2 Gene Family in Parnassius glacialis. Genes, 16(3), 249. https://doi.org/10.3390/genes16030249

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop